Abstract

Researchers interested in the heart have long used a wide range of imaging techniques to see, understand and quantify changes throughout heart development, repair and, in certain species, regeneration. Two major challenges in imaging the heart are the contrasting problems of high-frequency heart beating and low-frequency morphological changes. We have previously demonstrated how using prospective optical gating, in combination with light-sheet microscopy, can allow the synchronised capture of 3D images of the ınvivo beating zebrafish heart. However, prospective optical gating alone is limited to snapshots of the heart at chosen target heartbeat phases and only over the scale of tens of minutes. We have now developed hybrid prospective-retrospective optical gating technologies that we are using in combination with light-sheet microscopy to enable a range of 3D+time and 3D-timelapse imaging experiments. Here we will demonstrate several key areas where we have begun to exploit these technologies to further describe and understand cardiac function and dynamics. By incorporating these non-invasive optical gating methods with micro particle image velocimetry ($μ$PIV) we can achieve 3D+time resolved imaging of blood flow in the beating zebrafish heart. We use red blood cells as natural tracer particles from which we obtain instantaneous measurements of velocity from light-sheet images. Statistically combining and analysing this data, we can begin to both quantify and fully understand fluid-structure interaction in the developing zebrafish heart. Further, our hybrid prospective-retrospective optical gating technology allows us to carry out 24+ hour, ınvivo, 3D-timelapse imaging of the computationally `frozen’ heart across developmental stages, eg heart looping, and throughout injury response and repair. Imaging across these timescales is not possible with prospective optical gating alone and phase-locked timelapse imaging is not possible using retrospective optical gating alone: only with our hybrid system are such longitudinal studies possible. Our hybrid prospective-retrospective optical gating system allows researchers to study and understand cardiac development and repair without the use of chemicals or optogenetics to stop or modify the natural heart beating. Combining these powerful techniques produces a hybrid prospective-retrospective optical gating microscopy framework that we are now beginning to use to fully describe cardiac development, dynamics and repair as never before accomplished.